Our early crystal structures showed how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport. We expanded our studies in this area to determine how Neisseria meningitidis binds to human serum transferrin and extracts the iron for import into the bacterial cell. These bacteria require iron for survival and obtain it directly from human proteins. Neisseria have an outer membrane protein, TbpA, and a co-receptor protein, TbpB, which together can extract the iron from a human plasma protein called transferrin. We used a combined approach of X-ray crystallography, electron microscopy, small angle X-ray scattering, biochemistry, and molecular dynamics simulations to elucidate the iron-scavenging mechanism. This was the first atomic resolution structure of a bacterial outer membrane protein bound to its full-length human target protein. In our search for novel antimicrobial therapies, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the metal transporters that we study also facilitate the uptake of large protein toxins called colicins. For example, we determined the structure of an outer membrane iron transporter from Yersinia pestis (which causes plague) that is required for virulence. We also determined the structure of a colicin, called pesticin, which uses this transporter to cross the outer membrane. The two structures showed us how to engineer a novel antibiotic that is the first example of phage therapy for any Gram-negative bacterium, and our antibiotic was demonstrated to be effective on clinical isolates Guided by this success, we will continue this type of protein engineering for other bacterial pathogens. Interestingly, for all of these transition metal transporters, how the metal gets into the periplasm is not well understood. We know that transport involves an inner membrane protein complex (TonB-ExbB-ExbD) and energy in the form of protonmotive force. We recently determined the structure of a subcomplex of this motor, consisting of ExbB and ExbD. We used a combined approach of X-ray crystallography, electron microscopy, DEER spectroscopy, crosslinking, and electrophysiology to show that the Ton subcomplex forms pH sensitive, cation selective channels that couple ion flow to energy transduction at the outer membrane. Ongoing work 2018 Another hospital-acquired infection of great importance to the NIH clinical center is Klebsiella pneumoniae. This bacterium exhibits multidrug resistance and some strains have shown hypervirulence. In an effort to identify new ways to combat infection, we are collaborating with Susan Gottesman, NCI, to investigate proteins involved in regulation of capsule. K. pneumoniae can escape immune detection and prevent penetration of antibiotics with its thick capsule layer that surrounds the outer membrane. Our hypothesis is that down-regulation of capsule synthesis might make K. pneumoniae more sensitive to available antibiotics, and thus more treatable than is currently the case. Structural and functional experiments on this system are in progress. In a separate project targeting Klebsiella pneumoniae, we recently determined four structures of the Kp aerobactin transporter, which is a TonB dependent transporter that correlates with virulence in hypervirulent K. pneumoniae. We are currently using in silico drug screening and STD NMR to identify small molecules that compete for binding with aerobactin, with plans to explore these compounds in an animal model of the disease. This work will be finalized and published within the coming year. Working from our 2016 Nature publication on the TonB motor complex, we are currently exploring structure determination by cryo-EM to answer questions related to stoichiometry, subunit arrangement, and function. Our best data set is resolved to 5.5 A resolution and we are improving our samples for higher resolution data collection. We plan to publish a new structure within a year. References Buchanan, S.K., Smith, B.S., Venkatramani, L., Xia, D., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat. Struct. Biol. 6, 56-63. Yue, W.W., Grizot, S. & Buchanan, S.K. (2003). Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J. Mol. Biol. 332, 353-368. Buchanan, S.K., Lukacik, P., Grizot, S., Ghirlando, R., Ali, M.M.U., Barnard, T.J., Jakes, K.S., Kienker, P.K. & Esser, L. (2007). Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import. EMBO J. 26, 2594-2604. PMCID: PMC1868905 Noinaj, N., Easley, N.C., Oke, M., Mizuno, N., Gumbart, J., Boura, E., Steere, A., Zak, O., Aisen, P., Tajkhorshid, E.M., Evans, R., Gorringe, A., Mason, A.B., Steven, A. & Buchanan, S.K. (2012). Structural basis for iron piracy by pathogenic Neisseria. Nature 483, 53-58. PMCID: PMC3292680 Lukacik, P., Barnard, T.J., Keller, P.W., Chaturvedi, K., Seddiki, N., Fairman, J.W., Noinaj, N., Kirby, T.L., Henderson, J.P., Steven, A.C., Hinnebusch, B.J. & Buchanan, S.K. (2012). Structural engineering of a phage lysin that targets Gram-negative pathogens. Proc. Natl. Acad. Sci. USA, 109, 9857-9862. PMCID: PMC3382549 Mayclin, S.J., McCarthy, J.G., Botos, I., Lundquist, K., Majdalani, N., Wojtowicz, D., Barnard, T.J., Gumbart, J.C. & Buchanan, S.K. (2016). Structural and functional characterization of the LPS transporter LptDE from Gram-negative pathogens. Structure 24:965-76. PMCID: PMC4899211 Celia, H., Noinaj, N., Zakharov, S.D., Bordignon, E., Botos, I., Santamaria, M., Cramer, W.A., Lloubes, R. & Buchanan, S.K. (2016). Structural insight into the role of the Ton complex in energy transduction. Nature 538:60-65. PMCID: PMC5161667